Triple-zero tillage and system intensification lead to enhanced productivity, micronutrient biofortification and moisture-stress tolerance ability in chickpea in a pearlmillet-chickpea cropping system of semi-arid climate

Pearlmillet-chickpea cropping system (PCCS) is emerging as an important sequence in semi-arid regions of south-Asia owing to less water-requirement. However, chickpea (dry-season crop) faces comparatively acute soil moisture-deficit over pearlmillet (wet-season crop), limiting overall sustainability of PCCS. Hence, moisture-management (specifically in chickpea) and system intensification is highly essential for sustaining the PCCS in holistic manner. Since, conservation agriculture (CA) has emerged is an important climate-smart strategy to combat moisture-stress alongwith other production-vulnerabilities. Hence, current study comprised of three tillage systems in main-plots viz., Complete-CA with residue retention (CAc), Partial-CA without residue-retention (CAp), and Conventional-tillage (ConvTill) under three cropping systems in sub-plots viz., conventionally grown pearlmillet-chickpea cropping system (PCCS) alongwith two intensified systems i.e. pearlmillet-chickpea-fodder pearlmillet cropping system (PCFCS) and pearlmillet-chickpea-mungbean cropping system (PCMCS) in split-plot design. The investigation outcomes mainly focused on chickpea (dry-season crop) revealed that, on an average, there was a significant increase in chickpea grain yield under CAc to the tune of 27, 23.5 and 28.5% under PCCS, PCFCS and PCMCS, respectively over ConvTill. NPK uptake and micronutrient (Fe and Zn) biofortification in chickpea grains were again significantly higher under triple zero-tilled CAc plots with residue-retention; which was followed by triple zero-tilled CAp plots without residue-retention and the ConvTill plots. Likewise, CAc under PCMCS led to an increase in relative leaf water (RLW) content in chickpea by ~ 20.8% over ConvTill under PCCS, hence, ameliorating the moisture-stress effects. Interestingly, CA-management and system-intensification significantly enhanced the plant biochemical properties in chickpea viz., super-oxide dismutase, ascorbate peroxidase, catalase and glutathione reductase; thus, indicating their prime role in inducing moisture-stress tolerance ability in moisture-starved chickpea. Triple zero-tilled CAc plots also reduced the N2O fluxes in chickpea but with slightly higher CO2 emissions, however, curtailed the net GHG-emissions. Triple zero-tilled cropping systems (PCFCS and PCMCS) both under CAc and Cap led to a significant improvement in soil microbial population and soil enzymes activities (alkaline phosphatase, fluorescein diacetate, dehydrogenase). Overall, the PCCS system-intensification with mungbean (PCMCS) alongwith triple zero-tillage with residue-retention (CAc) may amply enhance the productivity, micronutrient biofortification and moisture-stress tolerance ability in chickpea besides propelling the ecological benefits under semi-arid agro-ecologies. However, the farmers should preserve a balance while adopting CAc or CAp where livestock equally competes for quality fodder.


Results and discussion
Soil microbial population. In current study, soil microbial population was significantly (p < 0.05) affected by various tillage practices and the intensified cropping systems. The colony forming unit (CFU) counts were significantly higher under complete conservation agriculture (CA) with residue retention (CA c ) followed by partial CA without residue retention (CA p ) treatments and conventional tillage (ConvTill), respectively (Fig. 1). Highest number of bacteria, fungi and actinomycete were seen under CA c followed by CA p . Across the tillage practices, pearlmillet-chickpea-mungbean cropping system (PCMCS) had significantly higher microbial CFU count followed by pearlmillet-chickpea cropping system (PCCS) while pearlmillet-chickpea-fodder pearlmillet cropping system (PCFCS) reported least microbial population. Among various treatment combinations, highest microbial counts of bacteria (82.2 × 10 4 CFU g −1 soil), fungi (63.2 × 10 2 CFU g −1 soil) and actinomycetes (49.5 × 10 4 CFU g −1 soil) were observed in CA c plots with PCMCS system (CA c _PCMCS) followed by CA c with PCCS system (CA c _PCCS) and CA c with PCFCS system (CA c _PCFCS), respectively. Lowest population of bacteria (53.3 × 10 4 CFU g −1 soil), fungi (41.5 × 10 2 CFU g −1 soil) and actinomycetes (29.3 × 10 4 CFU g −1 soil) were perceived under ConvTill_PCFCS which were 35.2, 52.2 and 16.2% lower than the best treatment combination CA c _PCMCS. This may be accrued to the reason the high organic biomass addition under CA c plots improved the soil structure, aggregate stability and uniform soil moisture availability, which in turn might have allowed microbial populations to grow and sustain in the rhizosphere [15][16][17][18]22 . Combining the CA c practice with legumeintensification also enhanced the SOC input owing to adequate leaf litterfall and root biomass additions from legumes with narrow C:N ratio 18,19 ; which in turn, enhanced the soil microbial diversity 17,[23][24][25] . Legume roots also release the root exudates which harbor the microbial diversity in the rhizosphere 4,20,26 , that's why the doublelegumes system i.e. PCMCS had highest microbial counts of bacteria, fungi and actenomycetes in current study. and alkaline phosphatase, glucosidase, dehydrogenase and fluorescein diacetate (FDA) activities (Fig. 2). These enzymatic activities were significantly (p < 0.05) higher under CA c followed by CA p , and ConvTill. Compared to ConvTill, the acid phosphatase, alkaline phosphatase, glucosidase, dehydrogenase and FDA activities were higher by 55.6, 64.3, 16.7, 105.3 and 83.8%, respectively under CA c . The system-intensification had significant effect on alkaline phosphatase, dehydrogenase and FDA activities. Highest activities of alkaline phosphatase (152 μmol p-nitrophenol g −1 h −1 ), dehydrogenase (454 μg TPF g −1 24 h −1 ) and FDA (24.2 μg fluorescein g −1 h −1 ) were observed under PCMCS, whereas PCFCS had least activities of these enzymes. As, legume-inclusion enhances the SOM 4,20,26 , thus, resulting in higher soil enzyme activities under double-legume system PCMCS 21,27 . Likewise, higher SOC enrichment both under CA-based tillage systems and the legume-intensification might have enhanced the FDA activity in our study 16,21 . The CA practices and legume-intervention also enhanced the dehydrogenase activity due to higher microbial nutrient bioavailability in the rhizosphere 16,28,29 . Crop productivity. Different Tillage practices and cropping systems had significant (p < 0.05) influence on the number of pods plant −1 during both years (Table 1). Pods plant −1 during 2020-2021 were 19% lesser than the year 2019-2020. Highest pods plant −1 (40.5 and 32.8) were obtained under CA c _PCMCS compared to rest of the treatments combinations during both years where ConvTill_PCFCS had least pod count plant −1 (31.4 and 26.6). Crop residue-retention improves the soil fertility and moisture holding capacity owing to SOM enrichment and nutrient bioavailability after biomass decomposition 13,21,30 , which accelerate the plant growth and dry matter accumulation and finally economic yield 31 . The CA practices are intended to increase carbon inputs, nutrient bioavailability with better physical rhizo-ecology (aggregate formation, moisture permeability and conservation) which directly proliferate the soil microbial diversity with higher crop yields 13,16 . Similarly, the interaction tillage and legume-inclusion (mungbean) showed significant (p < 0.05) grain and straw yield enhancement in chickpea during both years (Table 1). In general, chickpea grain and straw yield was comparatively higher during 2019-2020 than 2020-2021 owing to uniform rainfall distribution during 2019-2020 compared to 2020-2021 (Fig. 6). Significantly highest grain (1.23; 0.74 t ha −1 ) and straw yield (3.6; 2.06 t ha −1 ) of chickpea were recorded from the combination of CA c with PCMCS system over other combinations during 2019-2020 and 2020-2021, respectively. The CA c practice compared to ConvTill had a respective average grain yield increase by ~ 27, 23.5 and 28.5% and average straw yield increase by ~ 48.5, 47.5 and 56% under PCCS, PCFCS and PCMCS in our study. Again, the conventionally tilled PCFCS system had least grain and stover yield over other cropping sys-    Table 1. Interaction effect of tillage practices × cropping systems on pods plant −1 , grain and straw yields of chickpea. *Note: PCCS = Pearlmillet-chickpea cropping system; PCFCS = Pearl millet-chickpea-fodder pearlmillet cropping system; PCMCS = Pearlmillet-chickpea-mungbean cropping system; CA c = Complete conservation agriculture with residue retention; CA p = Partial conservation agriculture without residues; ConvTill = Conventional tillage.  www.nature.com/scientificreports/ tems. As, residue retention under CA plots was highly effective in reducing the evaporation losses and conserving more soil moisture, thus, resulting in better crop growth and yield over ConvTill plots 13,14 . Moreover, chickpea is a deep-rooted crop, therefore, which efficiently utilized the conserved soil moisture under CA plots for realizing higher yields 4,32 . There existed a significant positive and strong correlation between chickpea productivity and pods plant -1 during 2019-2020 (R 2 = 0.96) and 2020-2021 (R 2 = 0.77) (Fig. 3). The overall improvement in chickpea yield under CA plots (CA c and CA p ) could be ascribed to pivotal role of crop residues in several physiological, biochemical, chemical and physical processes 15-17,23,24,33 . Nutrient uptake. The experimental results revealed that both CA practices (CA c and CA p ) improved the total (grain + stover) NPK uptake in chickpea over conventional tillage (Table 2). Significantly (p < 0.05) higher total N (73.3 l; 43 kg ha −1 ), P (7.5; 4.3 kg ha −1 ) and K uptake ( Table 2. Interaction effect of tillage practices × cropping systems on total nutrient (N, P, K) uptake (kg ha −1 ) in chickpea. PCCS = Pearlmillet-chickpea cropping system; PCFCS = Pearl millet-chickpea-fodder pearlmillet cropping system; PCMCS = Pearlmillet-chickpea-mungbean cropping system; CA c = Complete conservation agriculture with residue retention; CA p = Partial conservation agriculture without residues; ConvTill = Conventional tillage.  www.nature.com/scientificreports/ conditions under CA plots could be the major factor for such observations 34 . Higher NPK uptake may also be accrued to higher yield under CA c owing to improved soil physico-chemical and biological properties 9,10 . Lowest NPK uptake was recorded from ConvTill_PCFCS owing to poor crop growth and biomass production in ConvTill plots compared to CAc 18,19,35 . Higher NPK uptake under PCMCS may also be accrued to inclusion of two legumes (chickpea and mungbean) in the system which greatly improved the soil biofertility over the PCCS and PCFCS systems 4 .
Micronutrient biofortification. Tillage practices and system-intensification had significant (p < 0.05) effect on micronutrient (Zn, Fe) biofortification in chickpea grains and straw (Table 3). Among tillage treatments, significantly (p < 0.05) greatest micronutrient content in chickpea grains as well as straw were obtained under CA c and CA p followed by ConvTill. The Fe and Zn content increased by ~ 2.5 and 1.56; and 8.3 and 10.1% in chickpea grains; and 3.4 and 3.8; and 3.7 and 6.2% in straw during 2019-2020 and 2020-2021, respectively over ConvTill. The improvement in micronutrient content under CA c may be attributed to enhanced microbial activity and synchronous nutrient release during SOM decomposition process of the crop residues 16,24,36,37 . Likewise, the highest micronutrient content (Zn, Fe) in chickpea grains and straw were observed under PCMCS owing to higher nutrient acquisition and biomass productivity under the influence of two legumes i.e. chickpea and mungbean 4 . Significant enhancement in micronutrients (2-years' av.) under different cropping systems was found to be 1.60 and 1.80 (Fe); 3.9 and 3.5 (Zn) mg kg −1 in grain and stover in PCCS and PCMCS, respectively over PCFCS. As, legume-imbedded systems fixed more N with sufficient biomass additions having narrow C: N ratio 18,19 ; thus, speeding-up the biomass decomposition with more C-sequestration vis-à-vis more micronutrient acquisition 4,27 . The resultant SOM might have also helped in synthesis of organic acids in rhizosphere 27 , which in turn, acted as micronutrient chelates, influencing translocation and remobilization of micronutrients 37,38 .
Relative water content. Various treatment combinations significantly (p < 0.05) improved the relative water content (RWC) in fully expanded chickpea leaves at flowering (Fig. 4). The highest RWC (86.3%) was achieved under CA c in PCMCS system. This treatment combination improved the RWC by ~ 20.76% over ConvTill_PCFCS system. The improved RWC under CA c was a consequence of higher moisture retention and comparatively lower moisture stress in residue-retained CA c plots 9,10 . As, the legume intervention in the crop sequences enhances the water holding capacity due to better physical and biological rhizospheric environment, hence, resulting in favorable plant-soil-water relations with higher RWC 18 Table 3. Effect of tillage practices and cropping systems on micronutrient (Fe, Zn) biofortification in chickpea grains. PCCS = Pearlmillet-chickpea cropping system; PCFCS = Pearl millet-chickpea-fodder pearlmillet cropping system; PCMCS = Pearlmillet-chickpea-mungbean cropping system; CA c = Complete conservation agriculture with residue retention; CA p = Partial conservation agriculture without residues; ConvTill = Conventional tillage.  www.nature.com/scientificreports/ Biochemical properties vis-à-vis moisture-stress tolerance ability. Tillage practices and systemintensification had significant (p < 0.05) influence on biochemical properties vis-à-vis moisture-stress tolerance ability of chickpea (Fig. 6), except ascorbate peroxidase (APX) and catalase (CAT) activity. Treatments, CA c _ PCMCS (22.9%), a combination of complete CA and double legume imbedded cropping system exhibited highest grain protein content (Fig. 6A), with ~ 3.6% higher protein content compared to ConvTill_PCCS. Higher N content in chickpea under CA c _PCMCS may be attributed to increased N-bioavailability in the soil due to double legume-inclusion 1,4,18 . Higher decomposition rate of crop residues in CAc system might have also enhanced the N-acquisition and protein content in the plants 18,39 . Grain protein content was least in ConvTill_PCFCS be due to extensive N removal by two cereal components in the system 9,10 . The proline content was found to be inversely related with RWC. The maxima of proline content (9.5 μmol g -1 FW) was obtained under Con-vTill_PCFCS (Fig. 6B). This treatment combination remained at par with ConvTill_PCCS (7.98 μmol g −1 FW) and ConvTill_PCMCS (6.84 μmol g −1 FW) and CAp_PCFCS (7.15 μmol g −1 FW). The least proline content was noticed in CAc_PCMCS and CAc_PCCS. The reduced proline levels in chickpea leaves in CA c might be due to the increased moisture retention under crop residues, which resulted in low plant moisture-stress 25,40 . Similarly, chickpea plants grown with CA practices in PCMCS showed significantly higher biochemical properties like superoxide dismutase (SOD) activity (28.9 Ug −1 FW −1 ), and glutathione reductase (GR) activity (0.63 U mg −1 protein −1 min −1 ) which were ~ 11 and 30% higher over ConvTill_PCCS (Fig. 6C,D). Statistically non-significant increase was noticed in the CAT and APX activities under CA c (Fig. 6E,F). Least proline and higher values of SOD, GR, CAT and APX activity in chickpea under CA c indicate the ability of CA-management on moisturestress tolerance in the current study 2,18 . It is evident from various studies that moisture or drought stress causes oxidative stress by decreasing stomatal conductivity in the plants which confines CO 2 influx in to the leaves 40 .
Hence, there is reduction in the leaf internal CO 2 , causing formation of reactive oxygen species (ROS) mainly in plant cell, mitochondria, chloroplasts and peroxisomes 41 . In our study, there was higher production of SOD, GR, CAT and APX activity in chickpea under CA c . As, higher ROS production induces deleterious impact on plant cells; the plant defense system becomes active against ROS 42 ; and releases non-enzymatic antioxidants (proline) and antioxidant enzymes (like CAT, SOD) and ascorbate-glutathione (AsA-GSH) cycle enzymes (like GR and APX) for detoxification of ROS and plant cell protection [40][41][42][43] . It indicates that enhanced SOD, GR, CAT and APX activities under CA c inducts drought-stress tolerance ability in chickpea plants in semi-arid environment.

GHG-emissions.
In current study, the CO 2 and N 2 O emissions ranged between 1757 and 2246 kg ha −1 and 332-345 kg ha −1 , respectively under various tillage treatments ( Table 4). The CAc system emitted relatively larger amount of CO 2 followed by CA p and ConvTill. The CA p and ConvTill remained statistically at par in terms of CO 2 emissions. Contrary to CO 2 , the N 2 O emission was the larger under ConvTill and lowest in CA c plots; however, net GHG-emissions were least under CA c compared to ConvTill. Likewise, the system-intensification (PCMCS and PCFCS) led to slight enhancement in CO 2 emissions both of which remained statistically at par. Intensive cropping (PCMCS/PCFCS) didn't affect N 2 O emissions where all the cropping systems behaved statistically similar. Zero-tillage with residue retention in intensive cropping systems increased the availability of organic carbon that might have resulted in enhanced soil respiration and CO 2 release 44,45 . Presence of residuecover reduces the N 2 O emissions 46 , and therefore, the slightly lower N 2 O flux was observed in the CA-systems 47 .

Conclusions
In current study, the triple zero-till based system-intensification coupled with residue retention (CA c ) may enhance the chickpea grain yield by ~ 25% over conventional tillage (ConvTill) systems in the moisture-starved semi-arid ecologies. Likewise, the double legume bound triple cropping system i.e. pearlmillet-chickpea-mungbean cropping system (PCMCS) under CA c significantly enhanced the relative leaf water content (~ 21%), total NPK uptake, protein content, micronutrient (Fe, Zn) biofortification, soil microbial population and soil enzyme activities compared to ConvTill. The micronutrient biofortification (Fe, Zn) in chickpea grains followed the trend of CAc > Cap > ConvTill. The N 2 O emissions remained unaffected under different cropping systems. Interestingly, the CA c -management reduced the N 2 O fluxes but with slightly higher CO 2 emissions, however, curtailing the net GHG-emissions. Triple cropping systems and the CA-management significantly influenced the plant biochemical entities in chickpea viz. proline content, super-oxide dismutase, ascorbate peroxidase, catalase and glutathione reductase. Least proline and higher values of superoxide dismutase, glutathione reductase, catalase and ascorbate peroxidase activity in chickpea under CA c indicate the ability of CA-management on moisturestress tolerance under semi-arid ecologies. Overall, the system-intensification of pearlmillet-chickpea cropping system by the mungbean (PCMCS) coupled with triple zero-tillage and residue-retention (CA c ) may enhance  (Fig. 7). The soil of the experiment was sandy loam in texture (Inceptisol), slightly alkaline in reaction, poor in soil organic carbon (SOC) and available-N and medium in available-P and available-K. Detailed initial physico-chemical properties of experimental soil are enlisted in Table 5.

Treatments detail and crop management.
The experiment was laid out in a split-plot design with three replications. In main-plots, tillage and residue management practices were used while diverse cropping systems were allotted in sub-plots (Table 6). Chickpea variety 'Pusa-1103' was sown in 2nd week of October during both years, at 45 cm row spacing using 80 kg seed ha −1 . Gap filling and thinning operations were done within 20 days of sowing. The crop was fertilized with 20 kg N + 40 kg P 2 O 5 + 40 kg K 2 O per ha. Plant nutrients; N, P and K were applied through urea (46% N), single superphosphate (16% P 2 O 5 ) and muriate of potash (60% K 2 O), respectively. The whole amount of NPK fertilizers were applied as basal at sowing of the chickpea. All the crops including chickpea were raised entirely under rainfed conditions on conserved soil moisture. To control weeds, pre-emergence application of pendimethalin was done using 0.75 kg a.i. ha −1 in 400 L ha −1 spray solution. Under CA plots, after harvest of preceding crop, paraquate 0.75 kg a.i. ha −1 was applied using 400 L water ha −1 as spray solution. All the crops were grown using standard package of practices except the respective treatment plans 51,56 .
Soil sampling and analysis. Current experimentation was done on a long-term experiment. Fresh and moist soil samples from 0 to 15 cm depth were collected immediately after completion of two years of crop rotations of the experiment conducted during 2019-2020 and 2020-2021 i.e. 3rd and 4th year of the long-term experiment. These samples were then transferred to the laboratory for microbial analysis. The total bacterial population was counted using the Pour plating method 57 , in which the samples were incubated on nutrient agar medium for 3 days at 32 °C. Counting of total fungi was performed after incubating the fungal culture plate at 30 °C for 5 days on rose bengal agar medium supplemented with streptomycin (30 µg ml −1 ) 58 . Likewise, total actinomycete counting was done using actinomycete isolation agar (AIA) plates with 50 mg ml −1 nalidixic acid 59 , where the AIA plates were incubated for 7 days at 28 °C. The results of triplicate readings were presented as CFU g −1 dry soil. The soil acid and alkaline phosphatase enzymatic activities were determined using 16 mM para (p)-nitrophenyl phosphate as substrate 60 and reported as μmol p-nitrophenol g −1 h −1 . Likewise, Glucosidase activity was estimated using 25 mM p-nitrophenol-β-D-glucopyranoside as substrate 61 and expressed as μmol p-nitrophenol g −1 h −1 . Dehydrogenase activity was determined by the rate of reduction of triphenyltetrazolium chloride to triphenylformazan 51 and expressed as μg TPF g −1 24 h −1 . Soil microbial activity expressed as FDA hydrolysis was determined following the method developed by Green et al. 62 .
Yield parameters and yield estimation. Pods plant −1 were counted from 10 randomly selected chickpea plants and there average was taken. Grain yield was recorded (at 14% moisture content) from the net plot area and expressed in t ha -1 following the methodology of Rana et al. 51 . Table 4. Effect of tillage practices and cropping systems on GHG-emission under pearlmillet-based cropping systems. *Note: PCCS = Pearlmillet-chickpea cropping system; PCFCS = Pearl millet-chickpea-fodder pearlmillet cropping system; PCMCS = Pearlmillet-chickpea-mungbean cropping system; CA c = Complete conservation agriculture with residue retention; CA p = Partial conservation agriculture without residues; ConvTill = Conventional tillage.  55 , and total P and K were determined using a sulfuric-nitric-perchloric acid digest 51 . Nutrient uptake was computed by multiplying respective nutrient Relative water content. Relative water content (RWC) of the chickpea leaf was determined from first fully expanded top leaf of the plants at flowering stage. Leaf fresh weight was recorded immediately and then the leaf was incubated in distilled water for at least 4 h at 40 °C in the dark, blotted dried and its turgid weight was measured. Finally, dry weight was determined after drying it at 80 °C for 48 h in the oven. The RWC was calculated with the following formula 63 : Here F w is the fresh weight, D w is the dry weight and T w is the turgid weight. (1)  www.nature.com/scientificreports/ GHG-emission studies. Fluxes of greenhouse gases (GHGs) i.e. CO 2 and N 2 O were measured during both chickpea growing seasons (October to March), using the static chamber method 70,71 and for continuous 7-days after fertilization and rainfall. Acrylic chambers of 15 cm × 15 cm × 100 cm size, fitted with thermometer, battery operated fan and rubber septa on the top were used for sampling of gases. Samples were collected once in a week between 9 and 11 AM using a 20 ml syringe fitted with a 3-way stopcock, at 0, 30, and 60 min after chamber closure. For each treatment, sampling was carried out in triplicate. CO 2 and N 2 O concentrations in the collected sample were analyzed by Gas Chromatograph (GC: Hewlett Packard 5890) 70 having a stainless steel column fitted with a flame ionization and electron capture detector. The cumulative amount of CO 2 and N 2 O emissions were determined by linear interpolation of two adjacent intervals of measurements carried out on the sampling days assuming that GHGs emissions followed a linear trend during the periods when no sample was taken 72,73 . The emissions of CO 2 and N 2 O from soil were calculated by the following equation: Here F is the CO 2 /N 2 O flux, ρ is the gas density, V is the volume of the close chamber (m 3 ), A is the surface area of the closed chamber (m 2 ), c t represents the rate of increase of CO 2 /N 2 O gas concentration in the chamber (mg/μg m −3 h −1 ) and T (absolute temperature) is calculated as 273 + mean temperature (°C) of the chamber. Total CO 2 /N 2 O flux for the entire cultivation period was computed by linear interpolation using the following Eq. 74 : where R i is the CO 2 /N 2 O emission flux (g m −2 d −1 ) on the ith sampling interval, D i represents the number of days in the ith sampling interval, and n is the number of sampling intervals.

Statistical analysis.
The data related to each parameter were analyzed as per the procedure of analysis of variance (ANOVA) to determine treatment effects through Tukey's honestly significant difference test as a post hoc mean separation test (p < 0.05) by using SAS 9.1 software (SAS Institute, Cary, NC). Tukey's procedure was used where ANOVA was found significant.

Research involving plants.
It is stated that the current experimental research on the plants comply with the relevant institutional, national, and international guidelines and legislation. It is also stated that the appropriate permissions has been taken wherever necessary, for collection of plant specimens. It is also stated that the authors comply with the 'IUCN Policy Statement on Research Involving Species at Risk of Extinction' and the 'Convention on the Trade in Endangered Species of Wild Fauna and Flora' .

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.